Alteration of the TALl gene is the most common genetic lesion associated with T cell acute lymphoblastic leukemia (T-ALL). Therefore, the TALl gene products are likely to be critical mediators of human leukemogenesis. Preliminary data indicate that TALl polypeptides have functional properties reminiscent of known transcriptional regulatory factors, including transactivation potential and sequence-specific DNA-binding activity. In common with other transcription factors, TALl may be subject to control by reversible protein phosphorylation. Therefore, the long-term goals of this project are to determine whether the leukemic properties of TALl are affected by phosphorylation, and, if so, whether the malignant potential of TALl can be mitigated by controlling its phosphorylation state. Preliminary results indicate that TALl is phosphorylated at three sites, represented by phosphopeptides l, 2, and 3. Therefore, our immediate goals are to identify the modified residues within these phosphopeptides and to examine the functional consequences of phosphorylation at these sites. We have already shown that phosphopeptide l is generated by modification of serine residue 122, a site within the transactivation domain of TALl. The other phosphorylated residues of TALl will be determined by a similar combination Of molecular techniques that includes site-directed mutagenesis, tryptic phosphopeptide mapping, and transient eucaryotic expression. Once the phosphorylated residues of TALl are defined, the effect of phosphorylation at each site will be evaluated with respect to three functional properties of TALl polypeptides - their transactivation potential, subcellular localization, and DNA-binding activity. The cellular enzymes that mediate TALl phosphorylation will then be identified by in vitro phosphorylation with purified protein kinases and by in vivo assays of protein interaction using the two-hybrid system. Identification of the kinases that directly modify TALl represents an early step towards our future goal of defining the signal transduction pathways that control the phosphorylation state of TALl in leukemic cells.